VITELLOGENIN SYNTHESIS IN FEMALE LARVAE OF THE GYPSY MOTH, L

Comp. Biochem. Physiol. Vol. 103B, No. 3, pp. 533-542, 1992 0305-0491/92 $5.00 + 0.00 Printed in Great Britain Pergamon Press Ltd

VITELLOGENIN SYNTHESIS IN FEMALE LARVAE OF THE GYPSY MOTH, L YMANTRIA DISPAR (L.):

SUPPRESSION BY JUVENILE HORMONE

HOWARD W. FESCEMYER,*t EDWARD P. MASLER,* ROmN E. DAVIS* and THOMAS J. KELLY* *Insect Neurobiology and Hormone Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705-2350, U.S.A.; and i'Clemson University, Department of Entomology,

! 14 Long Hall, Clemson, SC 29634-0365, U.S.A. (Tel. 803 656-3111; Fax 803 656-5065)

(Received 23 April 1992; accepted 29 May 1992)

Al~traet--l. Fat body from feeding-phase, last instar gypsy moth females incorporates L-[35S]methionine in vitro into two viteilogenins with the same molecular masses (165 and 180 kDa) as the apo-viteUogenins found in the hemolymph and the apo-vitellins in the eggs.

2. Both apo-viteUogenins are observed in the medium of fat body cultures, but only the 180 kDa apo-vitellogenin is observed in extracts of cultured tissue.

3. Synthesis and accumulation of the apo-vitellogenins are suppressed in a dose-dependent manner by topical treatment with the juvenile hormone analog, methoprene, prior to day 4.

4. This suppression suggests that a declining juvenile hormone titre is involved in the initiation of viteUogenin synthesis.

INTRODUCTION

Vitellogenins are the hemolymph precursors of the insect yolk protein, vitellin. In most insects, the initiation of vitellogenin synthesis in the fat body and/or the presence of vitellogenin in the hemolymph of adult females first occurs about the time that yolk deposition begins (Keeley, 1985; Postlethwait and Giorgi, 1985; Vafopoulou-Mandalos and Laufer, 1987). Although the precise endocrine pathways which control viteUogenin synthesis differ among insects, the initiation and/or maintenance of the synthesis of vitellogenin by the fat body of adult females is stimulated, either directly or indirectly, by the presence of juvenile hormone (Hagedorn and Kunkel, 1979; Engelmann, 1983, 1984; Hagedorn, 1985; Keeley, 1985; Vafopoulou-Mandalos and Laufer, 1987; Kumaran, 1990).

In the Lepidoptera, differences exist among species with regard to the timing of vitellogenin synthesis during development and the role of juvenile hormone in regulating vitellogenin synthesis (Hagedorn and Kunkel, 1979; Postlethwait and Giorgi, 1985). For example, vitellogenin synthesis by the fat body of Hyalophora cecropia (L.) first occurs in female larvae at the time of larval-pupal apolysis, which is well before yolk deposition (Pan, 1971). Allatectomy of diapausing H. cecropia pupae had no influence on vitellogenin synthesis (Pan, 1977). The appearance of vitellogenin in immature H. cecropia pupae or pupal adult intermediates was not influenced by allatectomy of fourth or early fifth instar larvae (Pan, 1977). Injection of juvenile hormone I had no influence on vitellogenin synthesis in isolated diapausing abdo- mens of H. cecropia pupae (Pan, 1977). In contrast, neither vitellogenin synthesis nor yolk deposition in Danaus plexippus (L.) are detected until after adult emergence. Vitellogenin synthesis in D. plexippus was prevented by neck-ligation of newly emerged adults

and restored by the injection of juvenile hormone I (Pan and Wyatt, 1976).

Mechanisms which regulate vitellogenin synthesis develop much earlier in females of the gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae), than in other insects. Apo-vitellogenin was detected in the hemolymph of 3-day-old, feeding-phase, last instar females (Davis et al., 1990b; Kelly et al., 1992). Using an ELISA method, Lamison et al. (1991) detected apo-vitellogenin in the hemolymph of 2-day- old last instars. The accumulation of apo-vitellogenin in the hemolymph was blocked by treatment of last instars with the juvenile hormone analog methoprene (Davis et al., 1990a; Kelly et al., 1992). Although vitellogenins have not been found naturally at such an early developmental stage in other insect species, juvenile hormone does suppress fat body synthesis of a number of non-vitellogenic, stage- specific proteins in at least four other lepidopteran larvae (Tojo et al., 1981; Jones et al., 1987; Ismail and Ray, 1988; Memmel and Kumaran, 1988). We report here on the role of juvenile hormone in regulating the in vitro synthesis of vitellogenin by the fat body of gypsy moth larvae and the in vivo accumulation of vitellogenin in the hemolymph.

MATERIALS AND METHODS Insect rearing and chemical treatment

Larvae used in all experiments were derived from the New Jersey strain of L. dispar (O'Dell et al., 1984). Handling of the eggs and preparation of the diet used to rear the post-embryonic stages were performed according to Bell et al. (1981). Post-embryonic stages were reared at 25_ l°C, 50-60% r.h. and on a photoperiodic regime of 16 hr light: 8 hr darkness. Larvae were reared through the fourth stadium at a density of 10/180 ml cup. Larvae designated as day one of the last (5th) instar were selected 5 hr after lights-on following eedysis during the previous lights-off

533

534 HOWARD W. FESCEMYER el al.

period. These larvae were reared at a density of 5/180 ml cup. Larvae designated as day - 1 of the last instar were fourth instars in the second day of head capsule slippage. Female larvae were used in all experiments. Segregation of the sexes by size dimorphism was performed during the fourth instar when females are larger than males.

The juvenile hormone analogs, racemic 7-R,S-methoprene and juvenile hormone I (Calbiochem, San Diego, CA), and the anti-juvenile hormone, racemic fluoromeval- onolactone (FMev; mol. w t= 148; D.C. Cerf, Zoecon- Sandoz, Palo Alto, CA), were dissolved in ethanol and topically applied (2 #1) to the dorsum of the abdomen. Although the day on which last instars were treated varied from - 1 to 4 (5 hr after lights-on), all treated larvae were used for hemolymph collection and/or in vitro fat body culture on day 5 (5 hr after lights-on).

Hemolymph collection and tissue dissection Larvae were anesthetized with CO2 before hemolymph

collection or tissue dissections were performed. Hemolymph was obtained by clipping a proleg and collecting 20#1 samples. Each sample was immediately diluted 1:6 with 4°C Bombyx saline (Okuda et al., 1985) containing 0.1% phenylthiourea (to inhibit tyrosinases). Hemocytes were removed by centrifugation at 12,000g and 4°C for 5 min. The supernatant was collected and stored at -90°C.

For tissue dissection, larvae were pinned ventral side down and the hemocoel was exposed by cutting the body wall along the entire dorsal midline. After removing the gut, the body cavity was rinsed with Bombyx saline. Perivis- ceral fat body tissue was then dissected from the hemocoel and washed for 5 rain in Bombyx saline. Dissection was performed at room temperature (ca 22°C).

Extraction of protein from tissue Previously frozen, cultured fat bodies were thawed on ice

and extracted with 4°C Bombyx saline (100 #1 final volume) which contained 10mM ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethylsulfonyl fluoride and no CaC12 . A plastic pestle that fit the incubation tube was used to homogenize the tissue. Homogenates were centrifuged at 12,000 g and 4°C for 20 min. The layer just above the pellet (middle layer) was designated as the tissue extract and stored at -90°C.

Determination of radioactivity incorporated into total protein Aliquots (5/~1) of the media and tissue extracts were

spotted onto discs of filter paper (1 cm diameter, Schleicher & Schuell No. 740-E) and allowed to air-dry (30 min). Protein was precipitated onto the discs according to the following method. Discs were soaked in 10% trichloroacetic acid (TCA) at 4°C for 10 min with gentle agitation, rinsed once with TCA at 4°C, then boiled for I0 min in TCA. Discs were then rinsed once with TCA at 4°C, soaked in TCA at 4°C for 10 min with gentle agitation, rinsed again with TCA at 4°C, rinsed once with ethanol at 4°C, air-dried for 30 rain, and immersed in a liquid scintillation cocktail (ACS ¢, Amersham). Discs without sample were included as back- ground controls. Aliquots (5 #1) of the media and tissue extracts were spotted on control discs which were used to determine radioactivity present before protein precipitation.

Statistical comparisons were made using a factorial design analyzed by the general linear models procedure of PC-SAS (SAS Institute, 1988). Probability (P) values in the Results section are for the independent type-III sums of squares F test. Student's t-test was used to determine if means were different from zero (Ho: /~ = 0).

In vitro culture of fat body Fat body was dissected and washed as described above.

Excess saline was removed by blotting the tissue on filter paper. The tissue was then placed into pre-weighed (accuracy to _0.01mg) polypropylene tubes (1.5ml, conical) containing 55/~1 Grace's medium without L-methionine (Gibco ®, Chagrin Falls, OH) which contained t #Ci L-[3SS]methionine (1222Ci/mmol, Amersham, Arlington Heights, IL) and unlabeled L-methionine where indicated. The tubes were weighed again to determine the wet weight of tissue (ca 10 mg) and the sealed tubes plus tissue were incubated for up to 12 hr at 26°C.

Following incubation, the culture was centrifuged at 12,000g and 4°C for 10min. As much of the incubation medium as possible was collected without disturbing the tissue pellet. To remove any remaining medium, the tissue was washed once with medium containing no L-[3SS] - methionine. This wash was discarded. Medium and tissue samples were stored separately at -90°C.

Electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophor-

esis (SDS-PAGE) was performed according to Laemmli (1970). Separating gels (12 x 14 cm; 1.5 mm thick) were cast as a linear gradient of 7-15% acrylamide. Proteins in the gels were detected by staining with Coomassie Blue or by fluorography (Fluor-Hance ~, Research Products Inter- national Corp., Mount Prospect, IL) enhanced autoradiography. The fluorographs were scanned using an Enhanced Laser Densitometer (LKB ULTROSCAN XLS). Prep- aration of partially purified egg vitellin was performed as described in Davis et al. (1990b).

RESULTS

Synthesis and release o f protein by cultured fat body Fat body from 5-day-old larvae incorporated the

highest level of L-[35S]methionine (P ~< 0.002) into

Table 1. Incorporation of L-[35S]methionine into total protein by fat body from 5-day-old, female gypsy moth larvae incubated for 6 hr in different media

Modifications to Incorporation* Grace's medium (cpm/mg tissue)

Oxygenf L-Methionine Medium Tissue extract - + 3392 ± 155a 5534 + 203b + + 2067 + 144c 4116 _ 138d - - 8459 _+ 214e 12,756 ± 225f + - 5285 + 202b 8619 + 188e

*Values are means±95% confidence intervals and means followed by the same letter are not significantly different (d = 0.05; Tukey's w procedure; N = 5/modification).

?A plus sign indicates that 02 was bubbled into the medium for 3 min and the air above the medium was evacuated with 02 before the tube was caped and sealed with modelling clay. A dash indicates that nothing was done to the medium or tube (oxygen levels were atmospheric).

Regulation of vitellogenin synthesis 535

i | v

0 " 3 " 6 " 9 " 12 ' ' ' 12 INCUBATION TIME (HR) INCUBATION TIME (HR)

Fig. I. Influence of incubation time and larval age on the/n vitro incorporation of L-[35S]methionine into the tissue (tissue extract) and medium (released) total protein by fat body from last instar, female gypsy moths. Numbers near each curve are larval age in days. Time 0 is fat body incubated for 5 rain. Points

represent means _+95% confidence intervals (N = 3 for days 3 and 7; N = 5 for day 5).

both tissue (tissue extract) total protein and medium (released) total protein when neither 02 nor cold L-methionine were added to the incubation environ- ment (Table 1). In each modification scheme, more label (P ~< 0.02) was present in tissue extract than in medium (Table l).

In vitro incorporation of L-[35S]methionine into tissue and medium total protein was maximum at 6 hr

for fat body from either 3-day-, 5-day- or 7-day-old larvae (Fig. 1). The amount of L-[35S]methionine incorporated into fat body total protein from larvae of all ages tested was greater (P ~< 0.035) than the amount released into the incubation medium. The amount of radioactivity in tissue and medium total protein for fat body incubated for 5 rain (time 0 in Fig. 1) was not different from zero (P > It I ~< 0.001).

M E D I U M F A T B O D Y T I S S U E 0 1 3 4 6 9 1 2 ~ = I N C U B A T I O N ~ . - O 1 3 4 6 9 1 2

T I M E ( h r )

- ~ a p o - V g 180J=- - ~ a p o - V g 165 ~,.-

7 0 - 8 0 k D " P R O T E I N ( s ) ='~

Fig. 2. Incorporation of L-[35S]methionine into tissue (tissue extract) and medium (released) proteins by fat body incubated/n vitro for up to 12 hr. Tissue was obtained from 5-day-old, last instar, female gypsy moths. Proteins were separated by SDS-PAGE and detected by fluorography enhanced autoradiography. Each lane contains 12,500 cpm of the total precipitated protein. Arrows indicate the positions of the 165 and 180 kDa apo-vitellogenins (apo-Vg) and presumed storage protein(s) (70-80 kDa proteins). These proteins were identified by comparing the fluorographs with the Coomassie stained position of these

proteins in lanes loaded with egg vitellin or hemolymph from 5-day-old, last instar, females.

CBPB 103/~2~

536 HOWARD W. FESCEM'YER et al.

Maximum incorporation of L-[35S]methionine into tissue and medium total protein was observed with fat body from 5-day-old, last instar females. Therefore, this larval age was used in all further experiments.

Fat body from 5-day-old, last instar, female larvae incorporated L-[35S]methionine into the 165 and 180 kDa apo-vitellogenins and presumed 70-80 kDa storage protein(s) (Fig. 2). The apo-vitellogenins were identified by comparing the fluorographs with the Coomassie-stained position of bands in lanes of the same gel loaded with egg vitellin, which contained the 165 and 180 kDa apo-vitellins, or hemolymph from 5-day-old, last instar, females which contained the 165 and 180 kDa apo-vitellogenins and 70-80kDa storage proteins (see the ETOH and egg vitellin lanes in Fig. 4a-e, for example). Densitometric scans of fluorographs, such as Fig. 2, showed that the incorporation of L-[3SS]methionine into the 165 and 180 kDa apo-vitellogenin band in the medium and the 180 kDa apo-vitellogenin band in the tissue increased with incubation time (Fig. 3). No 165 kDa apo-viteUogenin was observed in the tissue (Figs 2, 3), even though each lane of gels representing proteins in the medium or tissue was loaded with identical amounts of radioactivity. Accumulation of newly synthesized 165 kDa apo-vitellogenin in the medium increased with incubation time (Figs 2, 3), but was significantly (P ~< 0.022) less than the accumulation of newly synthesized 180 kDa apo-vitellogenin in the medium or tissue (Fig. 3). Incorporation of L- ['S]methionine into the 70-80 kDa proteins also increased with incubation time, as did other proteins (Fig. 2). Tissue was incubated for 6 hr in all further experiments because detectable levels of the 165 kDa apo-vitellogenin were observed only in the medium after 4--6 hr of incubation (Figs 2, 3).

Influence o f methoprene treatment Methoprene was topically applied once for each

age (days - 1 to 4 of the last instar) at doses ranging from 1 to 1000 nmol (0.31-310 pg/larva). Measure- ments were performed on day 5, and data on

hemolymph and fat body obtained from the same larva were analyzed with respect to day of treatment and dose applied.

Methoprene applied at 250 nmol or more on days - 1 , 1 or 2 blocked the accumulation of the 165 and 180 kDa apo-vitellogenins in the hemolymph by day 5 (Fig. 4a--c). These doses also blocked the incorporation of L-[35S]methionine into the 165 and 180 kDa apo-vitellogenins in the medium and the 180kDa apo-viteliogenin in the tissue extract of in vitro cultures of fat body from 5-day-old larvae (compare Fig. 5a-c with Fig. 6a--c).

Treatment of 3-day-old larvae with methoprene at 250 nmol or more suppressed, but did not block, the accumulation of the two apo-vitellogenins in the hemolymph by day 5 (Fig. 4d). These doses also suppressed the incorporation of L-[35S]methionine into the two apo-vitellogenins in the medium and the 180 kDa apo-vitellogenin in the tissue extract when fat body from 5-day-old larvae was cultured in vitro (compare Fig. 5d with Fig. 6a-c).

Methoprene treatment of 4-day-old larvae did not appear to change the accumulation of the apo-vitellogenins in the hemolymph by day 5 (Fig. 4e), or the incorporation of L-[3SS]methionine into the apo-vitellogenins when fat body from 5-day- old larvae was cultured in vitro (compare Fig. 5e with Fig. 6a-c).

Doses of methoprene from 1 to 1000 nmol did not appear to alter the accumulation in hemolymph of the 70-80 kDa storage proteins (Fig. 4a-e; heavy band above the 66 kDa standard). These same doses did not influence the in vitro incorporation of L-[3SS]methionine into the storage proteins by fat body from 5-day-old larvae (Fig. 5a-e).

In the in vitro cultures of day 5 fat body obtained from treated larvae, L-[sSS]methionine-labeled 165 kDa apo-vitellogenin was present only in the medium. In addition, no 165 kDa apo-vitellogenin band was detected in the tissue lanes by densitometric scanning of the fluorographs. L-[3sS]- methionine-labeled 180kDa apo-vitellogenin was present in both the tissue and medium (compare Fig. 5a-e with Fig. 6a-c). Moreover, the

4 A E E.a --z

2 P

G O.

I n c u b a t i o n T i m e (hr) Fig. 3. Influence of incubation time on the relative incorporation of u-[3SS]methionine into the 165 and 180 kDa apo-vitellogenins (apo-Vg) in the tissue (tissue extract) and medium (released) proteins by fat body/n vitro. Methods as in Fig. 2. Densitometric scanning of the fluorography enhanced autoradiographies represented by Fig. 2 was used to determine the relative amount of radiolabel (each lane contains 12,500 cpm of the total precipitated protein) in the 165 and 180 kDa apo-vitellogenin bands. Bars represent means +95% confidence intervals for three 5-day-old, last instar, females. A different gel electrophoresis

run was used for each larva.

npo-Vg180~ apo-VgleS~

Regulation of vitellogenin synthesis

A. TREATED ON DAY -1

METHOPRENE (nmole x 10) 1 ETOH 1 EGG VITELUN '

sro. ~.a..~.~o .L .U.4~. wo [ V r'*°ARDS

-~116kO "907kD ~SGkD

-R4SkD

~20kD

537

gpo-Vg180~ a'po'Vgle6~

B. TREATED ON DAY 1 EGG VITELLIN METHOPRENE (nmole x 10) T

Y STANDARDS ETOH2~ 1..! 10.ULaOZ.~. 199 Y !

~205kD

~116kD

~06kD ~45kD

~SgkD

C, TREATED ON DAY 2

METHOPRENE (nmole x 10) = ~GG VITELLIN

ETONO., , ,o . 50~,~o~TANOARDS aPo-Vg180~ -tgOSkD apo-Vg 165~ -~11SkO

-,ISTkO

",,eSkD

~4SkD

"qSSkD

,po-VglSO~. apo-VglSS~

D. TREATED ON DAY 3

METHOP.RENE (nmolo x 10) ETOHO.1 t 1Q . U . . k ~ S & l O 0 I GG VITELL~

~ TANDARDS

~2OSkD ~116k0 ~07kD

~66k0

~4SkD

~29kD

E. TREATED ON DAY 4 METHOPRENE EGG VITELLIN (nmole x 10) 8TANDARD8 s

STOH01 1 19..1280 7S *OO t v

IIpO-Vg 180~ |OSkO gpo-Vg 168 ~ 11 ekD

B7kD eekD t45kD

tSSkD

Fig. 4. Influence of methoprene, topically applied to different ages (indicated above each gel) of last instar, female gypsy moths, on the accumulation of protein in the hemolymph by day 5. Proteins were separated by SDS--PAGE and detected by Coomassie staining. Collected hemolymph was diluted 1 : 6 with Bombyx saline, and 5 pl of this dilution was loaded into each lane. Molecular weight standards (SDS-6H, Sigma Chemical Co., St Louis, Mo) are shown on the r i o t and the positions of the 165 and 180 kDa

apo-vitellogenins (apo-Vg) are shown on the left.

fluorographic densities of the L-[35S]methionine - labeled 180kDa apo-vitellogenin bands present in the medium were significantly (P ~<0.011) greater than those of the labeled 165 kDa apo-vitellogenin bands present in the tissue or medium (Fig. 6a-c).

Influence of juvenile hormone I and FMev treatment

Treatment of 2-day-old last instars with 20 nmol or more of juvenile hormone I suppressed the accumulation of the apo-vitellogenins in the

538

upo-V0180. spa-VOteS-

mpo-Protein~ 70-EOKD

HOWARD W. FESCEMYER et al.

A. T R E A T E D ON D A Y - 1

METHOPRENE (nmoie x 10)

E,o . . . . . do E,o. ~ , j 0 100 ~ ' ~ ~ '~r M T ~ ' ' ~ " " ~ ' T

aPo-Vg180~ aPo -Vo tQ6~

mDo-Prote~ ?O-80kD

B. T R E A T E D ON D A Y 1

METHOPRENE (nmo~e x 10) ErOH_0.,.k. ~ 1._~_0 2S ~ r._.~S ~00

M T M T M T Id T ~TT ~ T U T "~'~'r

apo-Vgt80~ s~o-V0188m

spo-Protein~ 70-00kD

C. T R E A T E D ON D A Y 2

METHOPRENE (nmole x 10)

ex°4~,.9..J- ~ d . 9 - _ ; . ! so loo

Ipo-VgleO~ aDO--VglO6~

tpo-Protoin~ ?O-80kD

D. T R E A T E D ON D A Y 3


ErOH O.1. . .L_I l o ~...~._5 5o r.//.5 lOO M._~T M T M T M T M T M T M T ~ 4 T

E. T R E A T E D ON D A Y 4


ETOH 0.1 1 10 25 50 75 100

10o-Vg180~ apo-Vgle5~

spo-Protein~ 70-80kD

Fig. 5. Influence of methoprene, topically applied to different ages (indicated above each gel) of last instar, female gypsy moths, on the incorporation of L-[35S]methionine into proteins present in the medium (M) and tissue (T) extract of in vitro cultures of fat body from day 5. The positions of the 165 and 180 kDa apo-vitellogenins (apo-Vg) and presumed storage proteins (apo-Protein 70--80 kDa) are shown on the left

and identified as described in Fig. 2. Methods as in Fig. 2.

hemolymph by day 5 (Fig. 7). The doses used (0 .01-200nmol; 0.29-588 #g/larva) did not appear to alter the accumulation in the hemolymph of the non-apo-vitellogenin proteins (Fig. 7).

FMev was topically applied once to ( - 2 ) - d a y - and l-day-old last instar larvae. Doses of 0 .1 -200nmol (0 .15-296#g/ larva) had no influence on accumulation of the 165 and 180kDa apo-vitellogenins or

Regulation of vitellogenin synthesis

A 180 kD a p o - V l t e l l o g e n l n B a n d In t h e M e d i u m a ~ . • T r e a t e d o n D a y - 1 i

4 [r ] , . . _ _ [ ] T rea ted on Day 1 ~ i ~ ~ T r e a t e d o n D a y 2 ]

° J i l l l ~ l L ~ . ~ Treated on D a y 3 1 - - ~ M U ~ ~ 4 ~ I T r . . t e d o n D a y 4 I

g. 0

ETOH 0 . 1 1 1 0 2 5 S 0 7 5 1 0 0 D o s e of M e t h o p r e n e ( n m o l e x 10)

539

B 165 kD a p o - V l t e l l o g e n l n B e n d a In t h e M e d i u m 3 ~ • Treated on Day -1 [

J [ [~ Treated on Day 1 [ I I _ _ T i [ ] T reated on Day 2 i

2~ ~ A-~ , ~ ~ - - [ ] T reated on D a y 3 [ t 1 3 • Treated on Day 4 [

ETOH 0 . 1 1 1 0 2 § 5 0 7 5 1 0 0 D o s e of M e t h o p r e n e ( n m o l e x 10)

C 180 kD a p o - V l t a l l o g e n l n B a n d In t h e T i s s u e 4-]1 1 Trea ted on Day-1 ]

s~ t h . • Treated on Day 1 t 3 "4 ~ T r=~ Trea ted on Day 2 [

J n d ; ~ gT! Treated on Day 3 [

"ll ,l'h " I 0 ETOH 0 :1 ; 1'0 2'5 5'0 7 '5 1 0 0

D o s e of M e t h o p r e n e ( n m o l e x 10)

Fig. 6. Influence of the methoprene treatment described in Fig. 5 on the relative incorporation of L-[35S]methionine into the 165 and 180 kDa apo-vitellogenin (apo-Vg) bands in the tissue (tissue extract) and medium (released) proteins from fat body incubations. Methods as in Fig. 2. Densitometric scanning of the fluorography enhanced autoradiographies represented by Fig. 5 was used to determine the relative amount of radiolabel (each lane contains 12,500 cpm of the total precipitated protein) in the 165 and 180 kDa apo-vitellogenin bands. Bars represent means +95% confidence intervals for three larvae. A

different gel electrophoresis run was used for each larva.

the non-apo-vitellogenins in the hemolymph by day 3 or 5. The FMev doses applied to 1-day-old larvae had no observable influence on development. All larvae treated with FMev on day-2 developed nor- mally, except for 10% treated with 100nmol and 60% treated with 200 nmol. These exceptions molted incompletely to the last instar and died.

DISCUSSION The suppression of in vitro vitellogenin synthesis,

by experimentally increasing the juvenile hormone level in female gypsy moth larvae prior to day 4 in the last instar, suggests that a low or declining hormone level is involved in the initiation of apo- vitellogenin synthesis during the first 3 days of the last instar. Once gypsy moth larvae begin apo- vitellogenin synthesis during days 2-3 in the last

instar (Davis et al., 1990b; Lamison et ai., 1991), methoprene treatment has a reduced influence on apo-vitellogenin synthesis.

These results demonstrate that synthesis of vitellogenin by the fat body of female gypsy moths differs from other insects in two important ways. First, vitellogenin synthesis was observed during the feeding-phase of the last instar. This is the earliest developmental stage of any insect species in which natural synthesis of vitellogenin has been docu- mented. The synthesis of vitellogenin in a specific, pre-imaginal stage, however, is consistent with observations on other Lepidoptera which develop mature eggs before adult emergence (H. cecropia, Pan, 1971; Manduca sexta L., Sroka and Gilbert, 1971; Bom- byx mori L., Kawaguchi and Doira, 1973; Pieris rapae L., Kim et aL, 1988). Second, elevated doses of juvenile hormone suppress vitellogenesis, suggest- ing that a decline in the juvenile hormone titre is

540 HOWARD W. FESCEMYER et al.

EGG V I T E L L I N JUVENILE HORMONE I (nmole) I S T A N D A R D S !

ETOH 0.010.1 1 10 20 5 0 1 0 0 2 0 0 ~ T

a p o - V g 1 8 0 ~ a p o - V g 1 6 5 v

a p o 7 P r o t e i ; = . . 0 - 8 0 k

~ 2 0 5 k D ~ 1 1 6 k D ~ 9 7 k D ~ 6 6 k D

~ 4 5 k D

~ 2 9 k D

Fig. 7. Influence of juvenile hormone I, topically applied to 2-day-old last instar, female gypsy moth larvae, on the accumulation of protein in the hemolymph by day 5. Methods as in Fig. 4. Molecular weight standards (SDS-6H, Sigma Chemical Co.) are shown on the right and the positions of the 165 and 180 kDa

apo-vitellogenins (apo-Vg) are shown on the left.

permissive for vitellogenin synthesis by the fat body of female gypsy moths. In contrast, vitellogenin synthesis by the fat body in most insect species which produce mature eggs as adults, occurs only when the level of juvenile hormone is increasing or already high (Hagedorn and Kunkel, 1979; Engelmann, 1983; Keeley, 1985; Vafopoulou-Mandalos and Laufer, 1987).

Methoprene and juvenile hormone I separately suppressed the accumulation of vitellogenin in the hemolymph (compare Figs 4c and 7), confirming that methoprene mimics the influence of the native hormone (Riddiford and Truman, 1978). Since topical application of FMev suppresses JH levels in M. sexta larvae (Baker et al., 1986), one may have expected FMev treatment to rapidly lower the level of juvenile hormone in ( -2)-day- or l-day-old last instar gypsy moths and thereby cause an early (i.e. on day 3) or increased (i.e. on day 5) accumulation of apo-vitellogenin in the hemolymph. However, FMev had no influence on the accumulation of apo-vitellogenin in the hemolymph. When compared to the results of Baker et al. (1986) for M. sexta, approximately 12 times more FMev is needed to produce an influence on gypsy moth larval development. Lepidopteran species vary in their susceptibility to the influence of FMev on larval development (Quistad et al., 1981; D. C. Cerf, personal communication), which suggests that FMev has little anti-juvenile hormone activity in gypsy moth larvae. Allatectomy may be the only alternative to FMev treatment, but allatectomy of "mature" (~<2500mg), last instar gypsy moths had no influence on the weight of ovaries in newly

emerged adults (Wang and Yin, 1983). The influence of allatectomy during the late fourth or early last instars on reproductive processes in the gypsy moth has not been examined. Occurrence of precocious metamorphosis and early initiation of vitellogenesis in aUatectomized larvae would suggest a close correlation between the initiation of vitellogenin synthesis and larval metamorphosis. The failure of methoprene treatment to block vitellogenin synthesis in 3- and 4-day-old last instar larvae suggests that, once initiated, vitellogenesis becomes refractory to methoprene treatment. Davis et al. (1990a) have examined the role, if any, of 20-hydroxyecdysone in the regulation of vitellogenin synthesis in gypsy moth larvae. They found 20-hydroxyecdysone to have no influence on vitellogenin synthesis in normal and methoprene-suppressed last instar females.

In a number of insect species, the responses of in vitro vitellogenin synthesis and accumulation to juvenile hormone treatment or aUatectomy have been shown to be correlated with the in vivo responses to juvenile hormone treatment or allatectomy (Hagedorn and Kunkel, 1979; Engelmann, 1983; Postlethwait and Giorgi, 1985). In L. dispar, methoprene treatment suppresses both the synthesis of the apo-vitellogenins in vitro and in the in vivo accumulation of the apo-vitellogenins in the hemolymph. The correlation of these in vitro and the in vivo data suggest that apo-vitellogenin synthesis in last instar gypsy moths is permitted at low juvenile hormone titers.

Newly synthesized, 165 kDa apo-vitellogenin was present only in the medium of fat body cultures.

Regulation of vitellogenin synthesis 541

Therefore, this protein is either rapidly released from the fat body subsequent to synthesis, or final modifications to the 165 kDa form occur outside the fat body. Post-translational processing of larger apo- vitellogenin peptides has been observed in other insect species, but the precise biochemical pathways are not well known (Postlethwait and Giorgi, 1985; Vafopoulou-Mandalos and Laufer, 1987; Wojchowski et al., 1986). Since the 180kDa apo- vitellogenin was present in the medium prior to the appearance of the 165kDa apo-vitellogenin, the 165kDa form may be derived from the 180kDa apo-vitellogenin.

In order to detect vitellogenin in the hemolymph or the medium and tissue extracts of fat body cultures, a relatively large amount of protein (ca 17/~g/lane) had to be loaded onto the gels. This resulted in the presence of a large amount of apo-protein in a band with a molecular weight ranging from 70 to 80 kDa. This molecular weight, synthesis by the fat body in vitro, and accumulation in the medium/n vitro, and in the hemolymph in vivo, all suggest that these apo- proteins are similar to the storage proteins detected in the hemolymph of other Lepidoptera (Levenbook, 1985). Although juvenile hormone regulates the synthesis of storage proteins by the fat body from some lepidopteran larvae (Tojo et aL, 1981; Jones et al., 1987; Ismail and Ray, 1988; Memmel and Kumaran, 1988), only the vitellogenin, synthesized by fat body from the last instar, was found to be regulated by juvenile hormone in L. dispar.

In the last instar of gypsy moths (Jones and Yin, 1989; Tanaka et al., 1989) and other Lepidoptera (Feyereisen, 1985; Riddiford, 1985; BoUenbacher, 1988), both the rate of/n vitro biosynthesis of juvenile hormone by corpora allata and the titre of juvenile hormone in hemolymph peak early in the feeding- phase (day 1 of this instar for corpora aUata activity in gypsy moths; Jones and Yin, 1989) and then decline over the course of the instar. Declines in both the synthesis of juvenile hormone by corpora allata and the titre of juvenile hormone in hemolymph are associated with the physiological events leading to larval-pupal metamorphosis (Riddiford, 1985; Bollenbacher, 1988). Our observations on juvenile hormone regulation of apo-vitellogenin synthesis correlate with those of Jones and Yin (1989) for corpora allata activity and those of Tanaka et al. (1989) for hemolymph juvenile hormone titre. It appears that the regulation of fat body vitellogenin synthesis in gypsy moth larvae is associated with endocrine events regulating larval-pupal metamorphosis.

Acknowledgements--We thank R. A. Bell for his assistance in rearing the insects and appreciate the critical suggestions of C. A. Sheppard, M. J. Loeb and D. Bolt. Mention of a commercial product does not constitute endorsement of that product by the USDA. H. W. Fescemyer was supported by USDA Competitive Grant No. 86-CRCR-1-2111 to E. P. Masler and T. J. Kelly.

REFERENCES

Baker F. C., Miller C. A., Tsai L. W., Jamieson G. C., Cerf D. C. and Schooley D. A. (1986) The effect of juvenoids, anti-juvenile hormone agents, and several

intermediates of juvenile hormone biosynthesis on the in vivo juvenile hormone levels in Manduca sexta larvae, Insect Biochem. 16, 741-747.

Bell R. A., Owens C. D., Shapiro M. and Tardiff J. R. (1981) Development of mass rearing technology. In The Gypsy Moth; Research Toward Integrated Pest Manage- ment (Edited by Doane C. C. and McManus M. L.), pp. 599-655. U.S. Dept. Agric. Tech. Bull. 1584.

Bollenbacher W. E. (1988) The interendocrine regulation of larval-pupal development in the tobacco hornworm, Manduca sexta: A model. J. Insect Physiol. 34, 941-947.

Davis R. E., Kelly T. J., Masler E. P., Fescemyer H. W., Thyagaraja B. S. and Borkovec A. B. (1990a) Hormonal control of vitellogenesis in the gypsy moth, Lymantria dispar (L.): Suppression of haemolymph vitellogenin by the juvenile hormone analogue, methoprene. J. Insect Physiol. 36, 231-238.

Davis R. E., Kelly T. J., Masler E. P., Thyagaraja B. S., Paramasivan V., Fescemyer H. W., Bell R. A. and Borkovec A. B. (1990b) Vitellogenesis in the gypsy moth, Lymantria dispar (L.): Characterization of hemolymph vitellogenin, ovarian weight, follicle growth and vitellin content. Invert. Reprod. Dev. 18, 137-145.

Engelmann F. (1983) Vitellogenesis controlled by juvenile hormone. In Endocrinology oflnsects (Edited by Downer R. G. H. and Laufer H.), pp. 259-270. Alan R. Liss, New York.

Engelmann F. (1984) Regulation of vitellogenesis in insects: The pleiotropic role of juvenile hormones. In Biosynthesis, Metabolism and Mode of Action of lnvertebrate Hormones (Edited by Hoffmann J. and Porchet M.), pp. 444-453. Springer, Berlin.

Feyereisen R. (1985) Regulation of juvenile hormone titer: Synthesis. In Comprehensive Insect Physiology, Biochem- istry and Pharmacology (Edited by Kerkut G. A. and Gilbert L. I.), VoL 7, pp. 391-429. Pergamon Press, New York.

Hagedom H. H. (1985) The role of ecdysteroids in repro- duction. In Comprehensive Insect Physiology, Biochem- istry and Pharmacology (Edited by Kerkut G. A. and Gilbert L. I.), Vol. 8, pp. 205-262. Pergamon Press, New York.

Hagedorn H. H. and Kunkel J. G. (1979) Vitellogenin and vitellin in insects. A. Rev. Entomol. 24, 475-505.

Ismall S. M. and Ray A. D.-G. (1988) Juvenile hormone inhibits the synthesis of major larval haemolymph proteins in rice moth, Corcyra cephalonica. Biochem. Intern. 17, 1093-1098.

Jones G., Hiremath S. T., Hellmann G. M., Wozniak M. and Rhoads R. E. (1987) Inhibitory and stimulatory control of developmentally regulated hemolymph proteins in Trichoplusia ni. In Molecular Entomology (Edited by Law J. H.), pp. 295-304. Alan R. Liss, New York.

Jones G. L. and Yin C.-M. (1989) Juvenile hormone biosynthesis by corpus cardiacum-corpus allatum complexes of larval Lymantria dispar. Comp. Biochem. Physiol. 92A, 9-14.

Kawaguchi Y. and Doira H. (1973) Gene-controlled incorporation of haemolymph protein into the ovaries of Bombyx mori. J. Insect Physiol. 19, 2083-2096.

Keeley L. L. (1985) Physiology and biochemistry of the fat body. In Comprehensive Insect Physiology Biochem- istry and Pharmacology (Edited by Kerkut G. A. and Gilbert L. I.), Vol. 3, pp. 211-248. Pergamon Press, New York.

Kelly T. J., Davis R. E., Fescemyer H. W., Thyagaraja B. S., Bell R. A. and Masler E. P. (1992) Regulation of gypsy moth vitellogenesis: A novel juvenile hormone suppression mechanism. In Proc. Fifth Int. Symp. on Juvenile Hormones (Edited by Mauchamp B.). INRA Press, Paris (in press).

Kim H. R., Ko Y. G. and Mayer R. T. (1988) Purifi- cation, characterization, and synthesis of vitellin from the

542 HOWARD W. FESCEMYER et al.

cabbage butterfly Pieris rapae L. Archs Insect Biochem. Physiol. 9, 67-79.

Kumaran A. K. (1990) Modes of action of juvenile hormones at cellular and molecular levels. In Morpho- genetic Hormones of Arthropods: Discoveries, Syntheses, Metabolism, Evolution, Modes of Action, and Techniques (Edited by Gupta A. P.), pp. 182--227. Rutgers University Press, New Brunswick, NJ.

Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage 1"4. Nature 227, 680-685.

Lamison C. D., Ballarino J. and Ma M. (1991) Tem- poral events of gypsy moth vitellogenesis and ovarian development. Physiol. Entomol. 16, 201-209.

Levenbook L. (1985) Insect storage proteins. In Comprehen- sive Insect Physiology Biochemistry and Pharmacology (Edited by Kerkut G. A. and Gilbert L. I.), Vol. 10, pp. 307-346. Pergamon Press, Oxford.

Memmel N. A. and Kumaran A. K. (1988) Role of ecdysteroids and juvenile hormone in regulation of larval haemolymph protein gene expression in Galleria mellonella. J. Insect Physiol. 34, 585-591.

Okuda M., Sakurai S. and Ohtaki T. (1985) Activity of the prothoracic gland and its sensitivity to prothoraci- cotropic hormone in the penultimate and last-larval instar of Bombyx mori. J. Insect Physiol. 31, 455-461.

O'Dell T. M., Bell R. A., Mastro V. C., Tanner J. A. and Kennedy L. F. (1984) Production of the gypsy moth, Lymantria dispar, for research and biological control. In Advances and Challenges in Insect Rearing (Edited by King E. G. and Leppla N. C.), pp. 156-166. USDA- ARS-SR, New Orleans.

Pan M. L. (1971) The synthesis of vitellogenin in the Cecropia silkworm. J. Insect Physiol. 17, 677-689.

Pan M. L. (1977) Juvenile hormone and vitellogenin synthesis in the Cecropia silkworm. Biol. Bull. 153, 336-345.

Pan M. L. and Wyatt G. R. (1976) Control of vitellogenin synthesis in the monarch butterfly by juvenile hormone. Devl, Biol. 54, 127-134.

Postlethwait J. H. and Giorgi F. (1985) Vitellogenesis in insects. In Developmental Biology: A Comprehensive

Synthesis (Edited by Browder L. W.), Vol. 1, pp. 85-126. Plenum Press, New York.

Quistad G. B., Cerf D. C., Schooley D. A. and Staal G. B. (1981) Fluoromevalonate acts as an inhibitor of insect juvenile hormone biosynthesis. Nature 289, 176-177.

Riddiford L. M. (1985) Hormone action at the cellular level. In Comprehensive Insect Physiology Biochemistry and Pharmacology (Edited by Kerkut G. A. and Gilbert L. I.), Vol. 8, pp. 37-84. Pergamon Press, New York.

Riddiford L. M. and Truman J. W. (1978) Biochemistry of insect hormones and insect growth regulators. In Biochemistry of Insects (Edited by Rockstein M.), pp. 307-357. Academic Press, New York.

SAS Institute (1988) SAS ~ User's Guide for Personal Computers: Statistics, Version 6.03. SAS Institute Inc., Cary, NC.

Sroka P. and Gilbert L. I. (1971) Studies on the endocrine control of post-emergence ovarian maturation in Manduca sexta. J. Insect Physiol. 17, 2409-2419.

Tanaka S., Chang M. T., Denlinger D. L. and Abdel-Aal Y. A. I. (1989) Developmental landmarks and the activity o f juvenile hormone and juvenile hormone esterase during the last stadium and pupa of Lymantria dispar. J. Insect Physiol. 35, 897-905.

Tojo S., Kiguchi K. and Kimura S. (1981) Hormonal control of storage protein synthesis and uptake by fat body in the silkworm, Bombyx mori. J. Insect Physiol. 27, 491-497.

Vafopoulou-Mandalos X. and Laufer H. (1987) Hor- monal regulation of fat body function in vitro. In Advances in Cell Culture (Edited by Maramorosch K.), Vol. 5, pp. 139-185. Academic Press, New York.

Wang Z.-S. and Yin C.-M. (1983) Larval allatectomy and reduced oviposition of the gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae). Ann. ent. Soc. Am. 76, 844-846.

Wojchowski D. M., Parsons P., Nordin J. H. and Kunkel J. G. (1986) Processing of pro-vitellogenin in insect fat body: A role for high-mannose oligosaccharide. Devl Biol. 116, 422 430.

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VITELLOGENIN SYNTHESIS IN FEMALE LARVAE OF THE GYPSY MOTH, L